Gram-negative bacteria can spontaneously release outer membrane vesicles (OMVs) during growth due to the turgor pressure of the cell envelope. The formation of such OMVs can be facilitated by disruption of certain bacterial components e.g. references 1 and 2 disrupted the E. coli Tol-Pal system to provide strains which release vesicles into the culture medium during growth. OMVs can also be produced by disruption of whole bacteria. Known OMV production methods include methods which use detergent treatment (e.g. with deoxycholate) [3 & 4], detergent-free methods [5], or sonication [6], etc.
OMVs are rich in immunogenic cell surface-associated, periplasmic and secreted antigens and have been used as vaccines, e.g. against Neisseria meningitidis serogroup B [7]. They are particularly suited for this use because the vesicles contain compounds that act as adjuvants, eliciting strong immune responses against the antigens. In this way, the vesicles are a closer mimic of the native bacterium for the immune system than purified antigenic proteins or other bacterial components. OMVs therefore remain an attractive target for vaccines and other immunogenic compositions. It has been suggested that the immunogenic properties of some protein antigens can be increased by engineering OMVs to display multiple antigens on the surfaces of OMVs by using ClyA as a fusion partner [8].
Several attempts have been made to target heterologous proteins, and in particular heterologous antigens, to OMVs. However, to date antigens that are foreign to the parental bacteria remain notably absent from OMVs largely because of challenges associated with the transport of heterologous proteins to the vesicles [11]. Most attempts to target heterologous proteins to OMVs have relied on covalent linkage of the heterologous proteins to integral membrane proteins. Examples of such covalently-linked heterologous proteins include fusions of the FLAG epitope to the full-length sequence of OmpA (outer membrane protein A), fusions of the FLAG epitope to the full-length sequence of PagP (PhoPQ-activated gene P) [9], and fusions of GFP to ClyA (Cytolysin) [10]. By virtue of their covalent linkages to membrane proteins, the resulting fusion proteins are targeted to the outer membrane and are thus included in the OMVs. These methods have drawbacks, in particular because it is difficult to overexpress a large amount of an integral membrane protein without detrimental effects of the transformed bacterium.
Targeting periplasmic proteins to OMVs has also proven to be difficult. A fusion of GFP to a Tat (twin arginine transporter) signal sequence resulted in overexpression of GFP that was targeted to the periplasm, but GFP fluorescence was barely above background fluorescence levels in OMVs [11], suggesting that the GFP was either not incorporated into the OMVs or was non-functional in the OMV because of incorrect folding.
There remains a need to develop a method suitable for expressing heterologous proteins in OMVs, and in particular a method to express antigenic proteins in OMVs. There also remains a need for alternative or improved OMVs, particularly for use in vaccines.